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PubMed2003

Patch-clamp recordings in isolated sponge cells (Axinella polypoides).

Sponges are the most ancient known metazoans. Their cells are specialised but not organised into tissues or organs. Recordings of action potential-like propagating electrical impulses suggested that electrical signalling may occur between sponge cells, but the characterization of ionic channels in these cells is still at the beginning. Actually, sponge cell surfaces are covered by a complex glycocalyx and long-chain fatty acids are present in the lipid core of their membranes. In these experimental conditions, a low percentage of tight seals (3%) was obtained applying the patch-clamp technique to cells isolated from the Mediterranean Demospongia Axinella polypoides. This paper shows in detail how difficulties can be overcome making use of trivalent cations in the extracellular solution and how electrophysiological measurements can be performed on sponge cell membranes. A potassium selective conductance is shown as an example. We suggest that the presented methodology could also be applied to other cell types.

Animals↗

PubMed1998

Detection of gene expression in single neurons by patch-clamp and single-cell reverse transcriptase polymerase chain reaction.

Detection and quantitation of gene expression in single cells is especially important in the central nervous system where, at the cellular level, the synapse can be considered the single functional unit. For example, the consolidation of long-term memories may be mediated by persistent changes in the strength of synaptic transmission at individual synapses. In order to investigate the requirement for de novo RNA synthesis during long-term potentiation in individual neurons, we have combined single-cell electrophysiology with single-cell gene-expression methodology. Described are methods combining whole-cell patch-clamp and single-cell RT-PCR for the detection of a single mRNA species for nitric oxide synthase, or, through a multiplex strategy, for the simultaneous detection of several mRNAs including heme oxygenase 2, protein phosphatase inhibitor 1 protein, and several isoforms of the calcium/calmodulin dependent protein kinase II.

Animals↗

PubMed2006

Whole-cell patch-clamp measurements of spermatozoa reveal an alkaline-activated Ca2+ channel.

In mammals, sperm cells become motile during ejaculation and swim up the female reproductive tract. Before fertilization and to overcome various barriers, their motility must be hyperactivated, a motion that is characterized by vigorous asymmetric tail beating. Hyperactivation requires an increase in calcium in the flagella, a process that probably involves plasmalemmal ion channels. Numerous attempts in the past two decades to understand sperm cell channels have been frustrated by the difficulty of measuring spermatozoan transmembrane ion currents. Here, by using a simple approach to patch-clamp spermatozoa and to characterize whole-spermatozoan currents, we describe a constitutively active flagellar calcium channel that is strongly potentiated by intracellular alkalinization. This current is not present in spermatozoa lacking the sperm-specific putative ion channel protein, CatSper1. This plasma membrane protein of the six transmembrane-spanning ion channel superfamily is specifically localized to the principal piece of the sperm tail and is required for sperm cell hyperactivation and male fertility. Our results identify CatSper1 as a component of the key flagellar calcium channel, and suggest that intracellular alkalinization potentiates CatSper current to increase intraflagellar calcium and induce sperm hyperactivation.

Alkalies↗

PubMed1995

A patch clamp study of the effects of ciprofloxacin and biphenyl acetic acid on rat hippocampal neurone GABAA and ionotropic glutamate receptors.

The neurotoxic effects of 4-quinolones alone and in combination with certain non-steroidal anti-inflammatory drugs (NSAIDs) may be related to an interaction at GABAA and/or ionotropic glutamate receptors. In the present study, the effects of the fluoroquinolone, ciprofloxacin, alone and in combination with the NSAID, biphenyl acetic acid (BPAA), were examined on GABAA-, NMDA-, AMPA-, and kainate-evoked current responses recorded from cultured rat hippocampal neurones, using the whole cell patch clamp technique. GABA-evoked currents were reversibly inhibited by bicuculline (3 microM) and ciprofloxacin (100 microM) to 11 +/- 5 and 38 +/- 7% of control, respectively. BPAA (100 microM) had little affect on the GABA current (the response was 82 +/- 4% of control) but enhanced the inhibitory potency of ciprofloxacin by approx. 3000-fold. The antagonist effects of ciprofloxacin (30 microM) and ciprofloxacin (0.03 microM) together with BPAA (100 microM) on the GABA-evoked current were not voltage-dependent. Whole cell currents evoked by NMDA, AMPA or kainate were little influenced by ciprofloxacin (100 microM), BPAA (100 microM), or ciprofloxacin plus BPAA (both at 100 microM); the responses being > or = 90% of control in all cases. These data suggest that the proconvulsant effects of quinolones when combined with BPAA may be related to antagonism of central GABAA receptors but not to an interaction at ionotropic glutamate receptors.

Animals↗

PubMed1995

Hormone-regulated Ca2+ channel in rat hepatocytes revealed by whole cell patch clamp.

An inward current responsible for hormone regulated Ca2+ entry has been identified in cultured rat hepatocytes using whole cell patch clamp. Addition of 20 nM vasopressin or of 100 microM ATP induced the inward current, which could be observed more clearly after blocking an outward K+ current. This large outward K+ current, which appeared after addition of vasopressin or ATP, could be blocked either by replacing K+ with Cs+ in the external medium and in the pipette solution, or by simply including 0.5 microM apamin in the K(+)-containing external medium. The outward current appears to be carried by a Ca2+ activated K+ channel. In the presence of apamin, hepatocytes pretreated with vasopressin in a Ca(2+)-free media reveal an inward current on addition of external Ca2+ (5 mM). The current could also be elicited by addition of vasopressin when cells are preincubated in the presence of 5 mM external Ca2+. No current is seen on addition of Ca2+ in the absence of vasopressin. Initially, the inward current was ca 200-300 pA at -60 mV, but it declined rapidly over 3 min to ca 20 pA. The current approached zero, as an asymptote at positive potential, and appeared to be somewhat inwardly rectifying. Additions of 5 mM Mn2+ or 5 mM Ba2+ in place of Ca2+ produced little or no current. An inhibitor of ER Ca(2+)-ATPase, thapsigargin, could also trigger the cascade of events leading to plasma membrane conductance of Ca2+. The data suggest that hormone-stimulated Ca2+ entry into hepatocytes is mediated by a Ca(2+)-release activated channel highly specific for Ca2+. This is the first demonstration of such a channel in hepatocytes, though similar ones have been described in mast cells, in vascular endothelial cells and T-lymphocytes.

Adenosine Triphosphate↗

PubMed1993

Blind patch-clamp recordings from substantia gelatinosa neurons in adult rat spinal cord slices: pharmacological properties of synaptic currents.

Whole cell patch-clamp recordings were made from substantia gelatinosa neurons in the thick slice of the adult rat spinal cord, which retained an attached dorsal root to study the pharmacological properties of spontaneous and primary afferent fibre-evoked synaptic currents. The majority of substantia gelatinosa neurons tested exhibited miniature excitatory postsynaptic currents in the presence of tetrodotoxin (0.5 microM). Stimulation of primary afferent A delta fibres evoked monosynaptic and/or polysynaptic excitatory postsynaptic currents. In Mg(2+)-containing solution, 6-cyano-7-nitroquinoxaline-2,3-dione (10 microM) abolished the evoked excitatory postsynaptic currents and the miniature excitatory postsynaptic currents. 2-Amino-5-phosphonovaleric acid (50-100 microM) had little effect on the miniature excitatory postsynaptic current. In Mg(2+)-free solution, however, 6-cyano-7-nitroquinoxaline-2,3-dione reduced but did not abolish the miniature excitatory postsynaptic currents, leaving the miniature excitatory postsynaptic currents with a small amplitude and a slow time course, which were abolished by 2-amino-5-phosphonovaleric acid. At holding potentials more positive than -60 mV, stimulation of A delta fibres evoked outward postsynaptic currents in 11 out of 28 substantia gelatinosa neurons. The evoked inhibitory postsynaptic currents were abolished in seven out of 11 neurons by either strychnine (0.5 microM) or bicuculline (10 microM), and in the remaining four neurons by the combination of both antagonists.(ABSTRACT TRUNCATED AT 250 WORDS)

2-Amino-5-phosphonovalerate↗

PubMed2004

Application of in vivo patch-clamp technique to pharmacological analysis of synaptic transmission in the CNS.

Slice preparations as well as acutely dissociated and cultured neurons from various regions in the CNS have been widely used to analyze pharmacological properties of synaptic responses and receptors expressed at the pre- and post-synaptic sites. However, the essential properties are not obviously different from neuron to neuron. These characteristics of neurons in the CNS make it difficult to elucidate their functional significances. It is, therefore, preferable that the pharmacological analysis should be made from identified neurons by stimulation of identified inputs. The in vivo patch-clamp recording technique allows us to clarify the synaptic responses evoked by the various known natural stimuli applied to the skin or other parts and makes it possible to interpret with more certainty the behavioral changes by synaptic plasticity observed at the single cell level. Although the in vivo technique has obvious advantages in analysis of physiological responses, this method is, however, confined so far to neurons located at the near surface of the CNS for pharmacological analysis, because of the diffusion problem of the chemical to deeper neurons. Thus, combinatorial studies with dissociated or cultured neurons or with slice preparations are clearly required for further understanding of pharmacological properties of neurons in the CNS.

Animals↗

PubMed2005

The interdependence and independence of amacrine cell dendrites: patch-clamp recordings and simulation studies on cultured GABAergic amacrine cells.

Previously we reported that cultured rat GABAergic amacrine cells can evoke subthreshold graded depolarization and action potentials. Both types of electrical signals are thought to contribute to neurotransmitter release from their dendrites, because Ca(2+) channels in amacrine cells can be activated at a subthreshold level (around -50 mV). The aim of the present study is to describe the spatiotemporal pattern of the spread of these electrical signals in an amacrine cell, using a computer simulation study. The simulation is based on physiological data, obtained by dual whole-cell patch-clamp recordings on the soma and the dendrites of cultured rat GABAergic amacrine cells. We determined passive and active properties of amacrine cells from the physiological recordings. Then, using the NEURON simulator, we conducted computer simulations on a reconstructed model of amacrine cells. We show that graded potentials and action potentials spread through amacrine cells with distinct patterns, and discuss the electrical interrelationship among the dendrites of an amacrine cell. Subthreshold graded potentials applied to a distal dendrite were sufficiently localized, so that each dendrite could behave independently (dendritic independence). However, at a suprathreshold level, once action potentials were triggered, they propagated into every dendrite, exciting the entire cell (dendritic interdependence). We also showed that GABAergic inhibitory inputs on the dendrites suppress the dendritic interdependence of amacrine cells. These results suggest that an inhibitory amacrine cell can mediate both local and wide-field lateral inhibition, regulated by the spatiotemporal pattern of excitatory and inhibitory synaptic inputs on its dendrites.

Action Potentials↗

PubMed1994

Comparative patch clamp studies on the kinetics and selectivity of glutamate receptor antagonism by 2,3-dihydroxy-6-nitro-7-sulfamoyl-benzo(F)quinoxaline (NBQX) and 1-(4-amino-phenyl)-4-methyl-7,8-methyl-endioxyl-5H-2,3-benzodiaze pine (GYKI 52466).

The glutamate antagonistic effects of NBQX [2,3-dihydroxy-6-nitro-7-sulfamoyl-benzo(F)quinoxaline] and GYKI 52466 [1-(4-amino-phenyl)-4-methyl-7,8-methyl-endioxyl-5H-2,3-benzodiaze pine] were compared on inward current responses of cultured superior collicular and hippocampal neurones with the whole cell patch clamp technique. Both NBQX (8 microM) and GYKI 52466 (33 microM) selectively reduced responses to AMPA [(S)-alpha-amino-3-hydroxy-5-methyl-4- isoxazolepropionic acid, 50 microM] and kainate (50 microM) whilst having little effect on responses to NMDA (N-methyl-D-aspartate, 100 microM). The effects of the two antagonists on the kinetics of AMPA (50 microM) responses were, however, very different--NBQX dramatically slowed the rise time of responses so that peak currents (IC50 60.4 +/- 4.2 nM) were markedly more effected than desensitized plateau currents (IC50 706 +/- 99 nM) whereas GYKI 52466 antagonized plateau responses (IC50 4.44 +/- 0.21 microM) somewhat more than peak responses (IC50 6.87 +/- 0.46 microM) and had only marginal effects on kinetics. In fact, low concentrations of NBQX (50-250 nM) actually potentiated plateau AMPA responses--an effect likely to be due to a reduction in the degree of AMPA-induced desensitization. Similar effects on response kinetics, were seen with kainate such that the IC50s for NBQX in antagonizing initial and plateau components of current responses to kainate 400 microM were 18.1 +/- 2.9 nM and 298 +/- 27 nM respectively whereas the IC50s for GYKI 52466 against kainate 50 microM were 17.3 +/- 1.8 microM and 15.5 +/- 3.3 microM respectively. These differences are likely to be due to the different modes of action of the two antagonists--NBQX shifted kainate concentration responses curves to the right in a parallel fashion indicative of competitive antagonism whereas the effects of GYKI 52466 were largely noncompetitive. There was, however, some indication for a small allosteric influence of GYKI 52466 on the affinity of the glutamate recognition site of the AMPA/kainate receptor. Estimation of Kbs using the Cheng-Prussoff relationship revealed little difference in the affinity of NBQX in antagonizing plateau responses to AMPA (Kb 23.2 nM) and kainate (Kb 57.1 nM) and indicate that the effects of these two agonists are mediated at a common receptor under the experimental conditions used. Moreover, the differential effects of NBQX on peak and plateau components of AMPA (50 microM) responses was associated with a desensitization-induced, paradoxical increase in the agonist affinity and was probably not due to any change in the affinity of NBQX.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

PubMed1999

Patch clamp studies on V-type ATPase of vacuolar membrane of haploid Saccharomyces cerevisiae. Preparation and utilization of a giant cell containing a giant vacuole.

A method for obtaining giant protoplasts of Escherichia coli (the spheroplast incubation (SI) method: Kuroda et al. (Kuroda, T., Okuda, N., Saitoh, N., Hiyama, T., Terasaki, Y., Anazawa, H., Hirata, A., Mogi, T., Kusaka, I., Tsuchiya, T., and Yabe, I. (1998) J. Biol. Chem. 273, 16897-16904) was adapted to haploid cells of Saccharomyces cerevisiae. The yeast cell grew to become as large as 20 micrometer in diameter and to contain an oversized vacuole inside. A patch clamp technique in the whole cell/vacuole recording mode was applied for the vacuole isolated by osmotic shock. At zero membrane potential, ATP induced a strong current (as high as 100 pA; specific activity, 0.1 pA/micrometer(2)) toward the inside of the vacuole. Bafilomycin A(1,) a specific inhibitor of the V-type ATPase, strongly inhibited the activity (K(i) = 10 nM). Complete inhibition at higher concentrations indicated that any other ATP-driven transport systems were not expressed under the present incubation conditions. This current was not observed in the vacuoles prepared from a mutant that disrupted a catalytic subunit of the V-type ATPase (RH105(Deltavma1::TRP)). The K(m) value for the ATP dose response of the current was 159 microM and the H(+)/ATP ratio estimated from the reversible potential of the V-I curve was 3.5 +/- 0.3. These values agreed well with those previously estimated by measuring the V-type ATPase activity biochemically. This method can potentially be applied to any type of ion channel, ion pump, and ion transporter in S. cerevisiae, and can also be used to investigate gene functions in various organisms by using yeast cells as hosts for homologous and heterogeneous expression systems.

Anti-Bacterial Agents↗

PubMed2004

An in vivo mouse spinal cord preparation for patch-clamp analysis of nociceptive processing.

The laboratory mouse is now considered the preferred mammalian species for molecular and genetic analysis in neurobiology. In part, this is due to the existence, in the mouse, of several well characterised naturally occurring mutations in ligand gated ion channels and recent knockout, knockin, and transgenic techniques, which facilitate the manipulation of key molecules. These techniques have recently been applied to pain research with in vitro electrophysiological and behavioural techniques traditionally developed for the rat, now being adapted for the mouse particularly at the level of the spinal cord. Here, we describe an in vivo preparation of the mouse spinal cord for patch-clamp recording of nociceptive processing in the superficial dorsal horn (SDH) that permits analysis in the intact nervous system. We have recorded from SDH neurons and characterised their background synaptic activity, discharge properties, and evoked synaptic responses following controlled application of innocuous and noxious stimuli to the hind paw. Application of these techniques along with genetic, biomolecular, in vitro and behavioural approaches will allow future studies to comprehensively analyse the contributions of specific molecules involved in nociceptive processing in the spinal cord of a single species.

Action Potentials↗

PubMed2003

Effects of Hypericum extract on the acetylcholine release: a loose patch clamp approach.

The St. John's Wort (Hypericum perforatum) extract (Hp) represents one of the most useful natural therapeutic agents in the treatment of moderate and mild depression. The antidepressant effects of Hp are different, by a molecular mechanism point of view, when compared to those of other antidepressant drugs and, we think, a further pharmacological characterization is needed. It is suggested that the neurochemical effects of Hp could be bind either to its activity on the uptake of some mediators in the central nervous system or to the inhibition of some enzymatic activity at the receptor level. The present study carried out with the loose patch clamp (LPC) in the mouse neuromuscular junction, indicates a potentiation of the acetylcholine (ACh) action at the mouse neuromuscular junction. The spontaneous release of ACh was unaffected by Hp indicating that neither presynaptic nor postsynaptic function are modified by Hp. Indeed, both the frequency and the amplitude of the miniature end-plate currents (mepcs) were unmodified by Hp. Furthermore, the mepcs decay time (tau), i.e. the apparent cholinergic channel life time, was significantly increased after Hp treatment. The other parameter affected was the amplitude of the evoked end-plate currents (epcs) which was constantly and in a dose dependent manner increased by Hp. These findings suggest a possible action of Hp on the acetylcholinesterase (AChE) in terms of a reduction of the degradation rate of ACh.

Acetylcholine↗

PubMed1998

Patch-clamp recordings from cerebellar basket cell bodies and their presynaptic terminals reveal an asymmetric distribution of voltage-gated potassium channels.

Cerebellar basket cells form highly specialized inhibitory synaptic contacts with Purkinje cells, namely the pericellular basket and pinceau nerve terminal structures, wrapping around the Purkinje cell somatic and axon hillock regions. These inhibitory synaptic contacts are ideally located to control the ultimate output of the cerebellar cortex. Previous immunohistochemical studies have shown that these synaptic structures possess a very high density of the dendrotoxin (DTX)-sensitive potassium channel subunit, Kv1.2. We have taken advantage of this unique anatomical arrangement offering a high concentration of identified Kv channel subunits by combining whole-cell patch-clamp recording and fluorescence microscopy to establish a novel preparation and perform the first recordings from unambiguously identified mammalian CNS inhibitory presynaptic terminals. We report that DTX-sensitive potassium channels are present in basket cell terminals but not in the basket cell soma. This selective cellular distribution suggests that these channels play an important role in modulating cerebellar inhibitory synaptic transmission.

Animals↗

PubMed1995

Patch clamp and atomic force microscopy demonstrate TATA-binding protein (TBP) interactions with the nuclear pore complex.

The universal TATA-binding protein, TBP, is an essential component of the multiprotein complex known as transcription factor IID (TFIID). This complex, which consists of TBP and TBP-associated factors (TAFs), is essential for RNA polymerase II-mediated transcription. The molecular size of human TBP (37.7 kD) is close to the passive diffusion limit along the transport channel of the nuclear pore complex (NPC). Therefore, the possibility exists that NPCs restrict TBP translocation to the nuclear interior. Here we show for the first time, with patch-clamp and atomic force microscopy (AFM), that NPCs regulate TBP movement into the nucleus and that TBP (10(-15)-10(-10)M) is capable of modifying NPC structure and function. The translocation of TBP was ATP-dependent and could be detected as a transient plugging of the NPC channels, with a concomitant transient reduction in single NPC channel conductance, gamma, to a negligible value. NPC unplugging was accompanied by permanent channel opening at concentrations greater than 250 pM. AFM images demonstrated that the TBP molecules attached to and accumulated on the NPC cytosolic side. NPC channel activity could be recorded for more than 48 hr. These observations suggest that three novel functions of TBP are: to stabilize NPC, to force the NPC channels into an open state, and to increase the number of functional channels. Since TBP is a major component of transcription, our observations are relevant to the understanding of the gene expression mechanisms underlying normal and pathological cell structure and function.

Animals↗

PubMed1995

Analogue computation of transient changes of intracellular free Ca2+ concentration with the low affinity Ca2+ indicator furaptra during whole-cell patch-clamp recording.

The measurement of cytosolic free Ca2+ ion concentration ([Ca2+]) with low affinity Ca2+ indicators has advantages for kinetic studies of cytosolic [Ca2+] transients when compared with more commonly used high affinity Ca2+ indicators. Their dynamic range and linearity are better suited to measurement of high localised transient concentration changes that exist near sites of influx or release, and the additional buffering introduced by the indicator is minimised. The fluorescent indicator furaptra (magfura-2) has low affinity for Ca2+ (approx. 50 microM) and can be used conveniently with single wavelength excitation at 420 nm with the procedure described by Konishi et al. [6]. The application of this protocol in whole-cell patch-clamp recording permits calibrated measurements of [Ca2+] during an experiment with minimal distortion of the time course and amplitude of [Ca2+] transients. A simple and inexpensive analogue circuit is described for direct computation of [Ca2+] from furaptra fluorescence with single wavelength excitation and emission during whole-cell recording. Data are shown which compare furaptra and fluo-3 estimates of the time course and amplitude of [Ca2+] changes in vascular endothelia, Purkinje neurones and hepatocytes.

Aniline Compounds↗

PubMed1994

Patch clamp analysis of the dominant plasma membrane K+ channel in root cell protoplasts of Plantago media L. Its significance for the P and K state.

Ion channels in the plasma membrane of root cell protoplasts of Plantago media L. were studied with the patch clamp technique in the cell-attached patch and outside-out patch configuration. An outward rectifying potassium channel was dominantly present in the plasma membrane. It appears responsible for the diffusional part, dominated by the K+ diffusion potential, of the cell membrane potential, in vivo. This channel is activated at potentials near to and more positive than the K+ diffusion potential. The dependence of this ion channel on K+ activity and voltage has been characterized. The current-voltage relationships of the open channel at various K+ concentrations are described by a four-state model. The membrane potential of intact protoplasts appears either dominated by the K+ diffusion potential, the protoplast is then said to be in the K state, or by the pump potential generated by the plasma membrane-bound proton pump/H+ ATPase, the P state. An experimental procedure is described to determine in cell-attached patch mode the state of the protoplast, either K or P state.

Cell Membrane↗

PubMed1995

Patch clamp detection of transcription factor translocation along the nuclear pore complex channel.

Transcription factors (TFs) are cytoplasmic proteins that play an essential role in gene expression. These proteins form multimers and this phenomenon is thought to be one of the mechanisms that regulate transcription. TF molecules reach their DNA binding sites through the large central channel of the nuclear pore complex (NPC). However, the NPC channel is known to restrict the translocation of molecules > or = 20-70 kD. Therefore, during their translocation, TF molecules and/or their multimers may plug the NPC channel and thus, interrupt ion flow through the channel, with a concomitant reduction in the ion conductance of the channel (gamma). Here we show with patch clamp that gamma is reduced during translocation of three major TFs: c-Jun (40 kD), NF-kappa B (approximately equal to 50 kD), and SP1 (approximately equal to 100 kD). Within a minute, femtomolar concentrations of these proteins reduced gamma suggesting a purely mechanical interaction between single TF molecules and the inner wall of the NPC channel. NPCs remained plugged for 0.5-3 hr in the absence of ATP but when ATP was added, channel plugging was shortened to < 5 min. After unplugging, channel closures were rarely observed and the number of functional channels increased. The transcription factors also stabilized the NPCs as shown by the extended duration of the preparations which allowed recordings for up to 72 hr. These observations are the first direct demonstration of the important role of NPCs in mediating nuclear translocation of TFs and, therefore, in forming part of the mechanisms regulating gene expression.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine Triphosphate↗

PubMed1998

Cloning, cDNA sequence analysis and patch clamp studies of a toxin from the venom of the armed spider (Phoneutria nigriventer).

The cDNAs (Tx3-2 and Pn3A) encoding precursor of toxin Tx3-2 and an isoform called Pn3A have been isolated from a library constructed from stimulated venom glands of the spider Phoneutria nigriventer. The cDNA of Tx3-2 reveals the presence of a signal peptide of 21 amino acids and of an intervening propeptide (with 16 amino acids) preceding the toxin sequence, which was followed by additional amino acid residues at the C-terminus (C-terminal peptide), implying post-translational modifications of the synthesised peptide. The deduced amino acid sequence for the mature toxin confirms the previous sequence published. In addition, by using the whole-cell patch clamp technique, we have determined that purified Tx3-2 decreases L-type currents present in GH3 cells. Finally, the presence of the cDNA Pn3A, with high sequence identity with Tx3-2, reveals the existence of a putative new toxin showing, at the cDNA level, 85.4% identity in its whole segment.

Amino Acid Sequence↗